Ultrasound imaging is the first-line investigation for patients with abdominal symptoms, as it effectively depicts the gastrointestinal tract and enables the diagnosis of multiple pathological conditions. Among different recent ultrasound technological advancements, elastography enables the evaluation of various tissue characteristics, such as neoplastic transformation or fibroinflammatory status. In recent years, ultrasound elastography has been utilized extensively for the study of liver diseases and in numerous other clinical settings, including gastrointestinal diseases. Current guidelines suggest the use of transabdominal ultrasound elastography to characterize bowel wall lesions, to assess gastrointestinal contractility, to diagnose and grade chronic pancreatitis; however, no specific indications are provided. In the present paper, we summarize the evidence concerning the application of different ultrasound elastography modalities in gastrointestinal non-liver diseases. Ultrasound (US) imaging is the first-line investigation for patients with abdominal symptoms, as it effectively depicts the gastrointestinal tract and enables the diagnosis of multiple pathological conditions . Recent σ is the stress applied, ε is the strain produced, and E is the elastic modulus or Young’s modulus. High Young’s modulus indicates that the applied stress results in less strain; consequently, the tissue has low elasticity (or high stiffness), and vice versa. When shear stress produces a shear strain tangential to the surface, elastic modulus is indicated as shear modulus (G) [Elasticity is a property of a tissue to resist deformation and revert to its original shape after being subjected to a stress. Neglecting the viscous component, elastic properties of a tissue can be described according to Hooke’s law (Equation (1)):ulus (G) .v) is approximatively 0.5:G is the shear modulus, sc is the shear wave speed, and Shear modulus is related to speed propagation of shear waves in a medium and to Young’s modulus. Equation (2) described this relationship under the assumption that for incompressible medium, the Poisson’s ratio . The results of SWE can be displayed as either shear wave speed (m/s) or shear or Young’s modulus (kPa) due to the relation expressed in Equation (2) ,7, but eIn pathological phenomena such as neoplastic transformation and fibroinflammatory status, the elastic properties of tissue change and elastography could theoretically be a reliable method for detecting and quantifying these changes ,10.After its development in the 1990s , early aCurrent guidelines suggest the use of transabdominal ultrasound elastography (US-E) to characterise bowel wall lesions, assess gastrointestinal contractility, diagnose and grade chronic pancreatitis (CP); however, no specific indications are provided ,15.In the present paper, we summarize the evidence concerning the application of different US-E modalities in gastrointestinal non-liver diseases.Few studies employed the US-E to evaluate the relationship between esophagogastric wall elasticity and motility, and only two case reports describe the US-E characteristics of gastric wall neoplasia.Real-time SE using the cardiovascular pulsation as pressure was used to count the change in stiffness (CS) of esophagogastric junction (EGJ) for ≥15 s in patients undergoing esophagogastroduodenoscopy. The number of CS was significantly lower in patients with reflux esophagitis compared to patients with non-reflux esophagitis and in multivariate analysis; the absence or presence of reflux esophagitis was the only significant factor related to CS. Furthermore, CS showed an area under the curve (AUC) of 0.84 for the diagnosis of reflux esophagitis. These results suggest that US-E might be useful to assess the EGJ function . In anotFurther studies are needed to establish the role of US-E in assessing the organic lesions and motility disorders of the upper gastrointestinal tract.Despite the methodological limits imposed by the normal anatomy and physiology of the lower gastrointestinal tract , one of US-E showed a practical usefulness also in the emergency setting of acute appendicitis, while no studies investigated the role of US-E in patients with other common acute intestinal disorders such as diverticulitis.The IBD are characterized by chronic intestinal wall inflammation caused by immunoinflammatory altered mechanisms . DespiteNormal stiffness of terminal ileum in 139 healthy volunteers was described by Zhao et al., using 2D-SWE. The mean 2D-SWE value was 1.08 ± 0.25 m/s (or 3.84 ± 1.84 kPa). Different physiological factors such as gender, age, body mass index (BMI), profundity and bowel wall thickness did not appear to significantly influence these values . Higher-As only the inflammatory form is medically treatable, the most investigated applications of US-E in IBD concern its ability to differentiate between inflammatory and fibrotic bowel lesions.In an ex vivo human study analysing 17 bowel segments resected for known or suspected IBD, Dillman et al., demonstrated that the p-SWE values were significantly higher in high-fibrosis segments compared to low-fibrosis ones, whereas there was no difference between high-inflammation and low-inflammation segments. Furthermore, the p-SWE results moderately correlated with the fibrosis score but not correlated with inflammation . In anotDifferent studies assessed the association between fibrosis and qualitative, semi-quantitative and quantitative US-E. A semi-quantitative SE-based score was lower for inflammatory stenosis than for fibrotic stenosis, indicating that fibrotic stenosis is significantly stiffer . The strRecent studies have expanded the evidence regarding the role of SWE in IBD assessment. In a prospective study including 35 patients undergoing resection within one week after US-E, the 2D-SWE results were significantly higher in severe fibrosis compared to moderate and mild fibrosis (23 ± 6.3 vs. 17.4 ± 3.8 and 14.4 ± 2.1 kPa). A cut-off value of 22.6 kPa distinguished severe from mild/moderate fibrosis with high accuracy . However, no differences were observed between consecutive inflammation grades . CompareSince inflammatory and fibrotic tissue have distinct vascularization, a combination of US techniques could further differentiate the two categories. Inflammatory and fibrotic bowel stenosis showed differences in both the 2D-SWE and contrast-enhanced US (CEUS) parameters  . In addip = 0.045 and 2.18 vs. 1.76 m/s, p = 0.032, respectively). Mayo subscore and C-reactive protein (CRP) levels were not significantly correlated with the p-SWE values within the colon segments, indicating that disease activity is not related to the SWE values [p = 0.007) and negatively correlated with clinical severity score [Only few studies, as far as we are aware, has examined patients with UC. Ishikawa et al., were the first to demonstrate alterations in SE related to colonoscopy findings and disease activity in UC . SubsequE values . Similarty score . In actiIn paediatric patients with CD, Fufezan et al., demonstrated that US-E findings were related to disease activity. A qualitative and semi-quantitative evaluation of 48 bowel segments based on the SE colour code and strain ratio was performed. Both methods showed a significant correlation with fat infiltration and disease activity markers . RecentlSeveral therapeutic alternatives are currently available for IBD. Among them, antitumour necrosis factor (TNF) or anti-interleukins is widely used in moderate-to-severe disease ,46. US-Ep = 0.009) and was significantly lower in patients who achieved mucosal healing  after 14 week of anti-TNF therapy compared to patients who did not achieve this outcome  [In a prospective study conducted by Orlando et al., the strain ratio at baseline and 14 and 52 weeks after starting anti-TNF therapy was evaluated. The strain ratio at baseline was significantly higher in patients who underwent surgery during the follow-up compared to the non-surgical group  . In cont = 0.03) . Further = 0.03) .Multiple studies demonstrated that US-E of appendix differs significantly between patients with acute appendicitis (AA) and healthy controls.p < 0.001). A cut-off of 12.5 kPa resulted in AUC, sensitivity and specificity values for predicting AA of 97%, 93% and 100%, respectively [p < 0.001). However, appendix diameter measured at conventional US showed an AUC comparable to the 2D-SWE values for distinguishing patients with AA from controls (0.996 vs. 0.998) [In a prospective study including 11 healthy volunteers and 41 patients with suspected AA, the 2D-SWE had higher results in definitive AA cases compared to patients with suspected but unconfirmed AA and controls  .Indeed, standard B-mode exhibits a remarkable ability to detect acute inflammatory processes affecting the appendix. In this setting, the real advantage of US-E may lie in its capacity to enhance the diagnostic accuracy of the B-mode when it does not reveal the typical features of appendicitis . In a prospective study by Kapoor et al., real-time SE correctly identified all 25 patients with AA, while the B-mode missed three cases . In anotLastly, a relationship between US-E and the severity of AA was demonstrated. A mild/moderate grade of AA assigned by SE demonstrated a high negative predictive value for phlegmonous and perforated appendicitis  .To the best of our knowledge, only one study analysed the difference in the SE values between colorectal adenocarcinoma and adenomas. In this study, Havre et al., prospectively enrolled patients with scheduled elective bowel resection. The strain ratio measurement and qualitative classification of lesions were performed. Adenocarcinomas were classified in increased tissue hardness categories and showed a strain ratio significantly higher than that in adenomas. However, no significant correlation was found between strain ratio and the grading of adenocarcinomas or in advanced T stages .Clinical studies regarding the use of US-E in pancreatic diseases are listed in Defining normal elastography values for the pancreatic parenchyma is challenging since, even when using the same method, there is a degree of variation among the healthy control populations presented across the studies. One of the largest healthy cohorts consisting in 210 subjects was presented by Xie et al. Normal pancreatic head and body’s p-SWE values were 1.18 ± 0.23 m/s and 1.20 ± 0.20 m/s, respectively, and were not related to age, gender, BMI, waist circumference and organ dimensions . In contn = 68) compared to healthy subjects (n = 52) and CP patients (n = 46) . Using a cut-off value of 2.2 m/s, the negative predictive value of p-SWE to distinguish AP from healthy controls and CP patients was 97.8%. Interestingly, five patients with AP had the p-SWE values higher than the upper detection limit, but different grey scale and colour scale scores, suggesting that the combination of two elastography techniques could improve the distinction between the inflamed tissue and tissue necrosis [n = 44) and healthy controls in the p-SWE results [p < 0.001) [Different studies investigated the ability of US-E to distinguish acute pancreatitis (AP) from normal pancreas or other pancreatic diseases. Mateen et al., found that the p-SWE values were higher in AP patients (necrosis . In cont results . Given t results . Similar< 0.001) .p = 0.000) but remained elevated after 1 month. Furthermore, 2D-SWE was not correlated with hospitalization and biochemical parameters [p = 0.151) [Given that universal normal or pathological elastography values have not been clearly determined, it would be highly interesting to investigate the relationship between the change in elastography values between the initial time and subsequent follow-up and prognostic outcomes. In a prospective study classifying AP according to Atlanta criteria, the 2D-SWE values in patients with mild AP trended to be higher than those in patients with severe AP (9.05 ± 1.44 vs. 8.61 ± 1.72 kPa); however, the difference was not significant. After clinical improvement, the 2D-SWE values decreased considerably compared to the initial value  . Furtherp = 0.001) and showed a positive correlation with the number of pain episodes, suggesting that fibrosis increases after each episode [Another important issue is to clarify if US-E can early detect the evolution of AP in CP patients. In patients with idiopathic recurrent AP and without endoscopic ultrasound (EUS) criteria of CP, the mean p-SWE value was significantly higher compared to that of healthy controls , the 2D-SWE values were significantly higher than those in healthy controls (n = 34)  and decreased over two weeks of steroids therapy more than the change in the pancreatic parenchyma size, proving to be a sensitive tool in monitoring response to therapy. Interestingly, the SW dispersion slope, which reflects the viscosity of a tissue and is related to necroinflammation more than fibrosis, was significantly different between patients with autoimmune pancreatitis and controls  [The US-E could be useful also to monitor response to therapy. In patients with type 1 autoimmune pancreatitis (= 0.011) .CP is characterised by the progressive replacement of pancreatic parenchyma with fibrotic tissue and the potential of progression to exocrine and endocrine insufficiency . As far p = 0.037), with an AUC of 0.71 for diagnosed CP. Furthermore, the p-SWE value correlated with the number of the EUS criteria for CP [p < 0.001) [Regarding the diagnostic value of US-E in CP, in a prospective study including patients with suspected CP (diagnosed with EUS), the p-SWE of the pancreatic body had significantly higher results in patients with the confirmed diagnosis of CP compared to patients without CP  . Notably< 0.001) . FurtherPancreatic steatosis (PS) is an emerging pathological entity that can contribute to the development and progression of pancreatic inflammatory and neoplastic disease, as well as endocrine systemic alteration and metabolic disorders . Therefop = 0.000). In addition, the 2D-SWE values correlated with BMI, waist circumference and serum triglycerides, progressively increased in B-mode classes of PS, and were significantly higher in patients with metabolic syndrome [p < 0.01) [As triglyceride accumulation modifies the viscoelastic properties of tissue, US-E may reflect these alterations; however, a comparison with histological data is not easily achievable. Recently, Sezgin et al., showed that the 2D-SWE values were significantly higher in PS patients compared to non-PS group  .p = 0.011) [Although the US-E has been shown to improve the diagnostic accuracy of the B-mode in determining the precise type of pancreatic lesions , contrad= 0.011) . Likewis= 0.011) .The US-E may also have a prognostic role in pancreatic cancer. Preliminary results of the study conducted by Kawada et al., demonstrated that strain ratio modification was related to response to therapy . Furtherp = 0.045). A cut-off value of 8 kPa showed an AUC of 0.69 with high specificity (90%) and low sensitivity (57.1%) and categorized the 2D-SWE value as the only factor significantly related to acute cholecystitis [By using conventional US, acute cholecystitis is diagnosed with 81% sensitivity and 80% specificity [cystitis .p = 0.01) [US-E could be an additional tool to distinguish malignancies from benign condition of gallbladder wall. Using SE, gallbladder polyps were characterized by medium-high strain (soft or moderately soft) compared to only one single small nodule (measuring 19 mm) defined as gallbladder carcinoma which exhibited low strain (high stiffness) . Conside = 0.01) .In this review, we examined the numerous applications of elastography in a wide range of gastrointestinal disorders other than the well-established use in liver diseases. Elastography could be a useful adjunct to conventional US in an emergency setting. Elastography has the potential to be an efficient non-invasive diagnostic and characterization tool for acute and chronic conditions of the bowel, pancreas and gallbladder, and finally, it could be an easily accessible and low-cost tool for assessing or monitoring the response to specific treatments. On the other hand, the limitations of current research and the resulting inability to provide detailed indications from international guidelines prevent the application of elastography in routine clinical practise. Additional research is required to standardize the methodology of this technique and to understand the real importance of US-E in all the illustrated conditions.


The functionalizationwith a hydrophilic and soft polymer modifies the hydrophobicity ofgraphene and might enhance its biocompatibility. We have characterizedthese hybrid materials by atomic force microscopy (AFM), X-Ray photoelectronspectroscopy (XPS) and Raman spectroscopy and studied their electricalresponse, confirming that the graphene/p-AP/PHEAarchitecture is anchored covalently by the sp3 hybridizationand controlled polymerization reaction on graphene, retaining itssuitable electronic properties. Among all the possibilities, we assessthe proof of concept of this graphene-based hybrid platform as a humiditysensor. An enhanced sensitivity is obtained in comparison with pristinegraphene and related materials. This functional nanoarchitecture andthe two-step strategy open up future potential applications in sensors,biomaterials, or biotechnology fields.The modificationof the surface properties of graphene with polymersprovides a method for expanding its scope into new applications asa hybrid material. Unfortunately, the chemical inertness of graphenehinders the covalent functionalization required to build them up.Developing new strategies to enhance the graphene chemical activityfor efficient and stable functionalization, while preserving its electronicproperties, is a major challenge. We here devise a covalent functionalizationmethod that is clean, reproducible, scalable, and technologicallyrelevant for the synthesis of a large-scale, substrate-supported graphene–polymerhybrid material. In a first step, hydrogen-assisted plasma activationof  Otherwise, PA dissociation hasbeen limited to mono-atomic functionalization,20 but these limitations can be overcome if PA low-vacuumtechniques are used to promote the dissociation of organic moleculesin gas phase that immediately link to the graphene lattice. Thus,PA is a promising strategy for the covalent functionalization of graphene,as we address in this work. In addition, the PA low-vacuum processis fast, clean, cost-effective, and scalable.Graphene, an sp28 It is also a promising polymer with soft and hydrophilic propertiesand high water adsorption capacity,30 one of thekey factors for biocompatible and bioengineering applications. Therefore,we expect this hybrid material to be a great candidate to study themodification of the graphene hydrophobicity and its improvement inthe biocompatible behavior as in other cases where a polymer is combinedwith other materials.28Acrylate polymersare a type of significant vinyl polymers usedin a wide range of industrial products, ranging from contact lensesor bone cement to cosmetics, coatings, adhesives orthopedics, textilesor drug administration. Acrylates in combination with other components form interesting newhybrid materials for bioengineering. PHEA is a polymer broadly usedas a hydrogel for lens, hemotherapy, in bioactive scaffolds, controlledrelease and applications that prove its biocompatible character.p-aminophenol(p-AP) and the carrier H2 gas dissociatein a low-vacuum plasma chamber and the graphene layer is activated,enabling the covalent functionalization of the basal plane with theorganic moiety. We functionalize the substrate-supported graphenewith organic molecules in a low-vacuum clean plasma environment, asan unexplored alternative to the wet chemistry methods5 and UHV8 approaches. This (p-AP) organic molecule incorporates an end-group (phenol)which is intended to be the linker to further anchor more complexmolecules and subsequently obtain a hybrid material. The key advantageof this strategy is the versatility of the method that enables touse specific linker molecules to functionalize graphene on demand.18 Recently, a similar platform has been demonstrated,especially suitable for high-performance bioelectronic devices.31Here, we devise a new versatiletwo-stage functionalization approachto build large-area graphene–polymer hybrid nanoarchitecturesas platforms with potential applications in biotechnology and sensing.In a first stage, the organic linker molecule 32 from the OH group provided bythe p-AP molecule, resulting in a robust and stablegraphene/p-AP/PHEA hybrid functional material. Ourmethod keeps the suitable properties of functionalized graphene, sinceno solvents or high temperatures that could lead to the material degradationare used enhancing, in contrast, the versatility of graphene.36 This approach is highly versatile and opens the door to the combinationof a wide range of linkers and polymers with potential applicationsin several fields.41 For instance, graphene is a material with a large potential in high-performancehumidity sensors, as reported in the literature.44 Therefore,the combination of the graphene sensitivity, the robustness of thecovalent functionalization, and the water absorption capacity of thepolymer make the hybrid platform proposed as a potential candidateto be an efficient humidity sensor. Accordingly, we show in this worka proof of concept of such application.Afterwards, we carry out an in situ surfacepolymerization of 2-hydroxyethylacrylate (HEA) monomer that grows in a controlled and reproduciblemannerp-AP/PHEA architecture is anchoredcovalently, in a stable and robust way, by sp3 hybridizationof graphene bonds and controlled polymerization. Finally, we assessthe sensitivity of our graphene–polymer hybrid platform developedby measuring the surface electrical properties by the four-point probeanalysis after different relative humidity exposures as a proof ofconcept of a humidity sensor. This study describes a new versatileand reproducible route to design functional robust nanoarchitecturesbased on graphene that have shown breakthrough applications in differentfields.The topographic changesin graphene are sequentially characterizedby atomic force microscopy (AFM), and the chemical functionalizationis analyzed by X-ray spectroscopy (XPS) and Raman spectroscopy inboth graphite surfaces and supported graphene samples. We ascertainthat the graphene/22.12.1.145 During functionalization,the chamber pressure is 5.4 × 10–2 mbar, andthe plasma power is 200 W. HOPG purchased from Tip Nano OÚand Si/SiOx wafer-supported CVD graphene purchased from GrapheneaInc. are used as substrates. The p-aminophenol (p-AP) powder precursor is purchased from Sigma-Aldrich (H2NC6H4OH ≥ 98%), and H2 is used as a gas carrier. The second stage of functionalizationand the final step to build up the hybrid material is carried outin an oven with a constant nitrogen flow. The monomer and initiator,2-hydroxyethyl acrylate (HEA)  and 2,2-azobis(isobutyronitrile)(AIBN), the reactants used to carry out in situ free-radical (FR)polymerization on the surface, are purchased and used as receivedfrom Sigma-Aldrich.Thefirst stage of covalent graphene functionalization is carried outby using an ASTEX AX 4500 electron cyclotron resonance (ECR) plasma-assistedequipment. A comprehensive description of the plasma system can befound elsewhere.2.1.2Room-temperatureAFM measurements for topographical analysis are performed with twocommercial equipment: (a) Nanoscope IIIa from Veeco (United States):Dynamic operation mode is selected, and silicon cantilevers (Bruker)with a nominal radius of curvature of 8 nm and nominal constant forceof 1–5 N/m are used. (b) Instrument and software from NanotecElectrónica S.L: Dynamic operation mode is employed, excitingthe tip at its resonance frequency (∼75 kHz) to acquire topographicinformation of the samples. The structure of our graphene films isaddressed by Raman spectroscopy using a confocal Raman microscope(S&I Monovista CRS+). Raman spectra have been obtained using a532 nm excitation laser, a 100× objective lens (NA = 0.9), andan incident laser power of 6 mW. The chemical nature of the hybridmaterial is determined by XPS. XPS measurements are carried out underUHV conditions using a PHOIBOS 100 1D delay line detector electron/ionanalyzer, monochromatic Al Kα anode (1486.6 eV), and pass energyof 30 eV.2.1.3–6 mbar). The 94 ± 3% RH data were acquired after2 h of continuous exposure to a humidity-saturated environment usinga wet paper in an airtight Petri dish . The room RH (27 ± 3%) correspondsto the laboratory ambient conditions. Measurements correspond to I–V curves in a four-point probesystem JANDEL RMS2 Universal Probe with continuous current (from 0.1to 10 μA). We apply the standard equation R□ = 4.53·ΔV/I for sheet resistance (R□) calculations (ΔV being the change in voltagemeasured between the inner probes and I the currentapplied between the outer probes).The water vapor absorption ability onto a sample surfacebecauseof the polymer presence allows using the hybrid material as a humiditysensor that is assessed at 0% relative humidity (RH), room RH (27± 3%), and top RH (94 ± 3%). The 0% RH data were acquiredafter the samples were placed 2 h in a vacuum environment (102.2p-aminophenol (p-AP) and the 2-hydroxyethyl acrylate (HEA) in situ polymerizationon the surface.The build-up strategy includestwo main stages: the linker attachment consisting of a covalent functionalizationwith the organic molecule 2.2.1p-AP precursor powder isevaporated into the chamber at 100 °C during 10 min, while thegraphene or HOPG samples are at room temperature. After the p-AP gas pressure is stabilized, we introduce the H2 gas carrier and switch on the plasma during 30 s . The plasma activationdissociates atomic H and the organic precursor in radicals, as depictedin 3 soft hybridization of the basal plane.47 Note that this ECR plasma produces around 5–10% atomic H.This process enables the attachment of the p-AP organicradicals previously activated by plasma. In this way, a high densityof functionalization can be achieved, controlling time and dose . One plausible reaction path is theplasma-assisted dehydrogenation48 or dissociation49 of the amino group in plasma, leaving behindaromatic groups with hydroxide moieties that anchor to the buckledgraphene lattice, as pointed out by our XPS results below. We alsotested the attachment of the molecule by carrying out an ethanol washand taking AFM images before and after the process. We observe thatthe molecule remains intact . As a result, the phenol end-groupbecomes the linking moiety to anchor more complex molecules.2.2.22 flow in order to perform a slow andcontrolled polymerization based on previous kinetic studies reportedin the bibliography.32 We are interested in a slow polymerizationrate to obtain a low surface coverage. In this work, two in situ polymerizationsare carried out where one sample aims to have the lowest amount ofpolymer on the surface (L-PHEA) and the other intends to achieve higherpolymer coverage on graphene (H-PHEA). For L-PHEA, the reaction timeis 30 min, while for H-PHEA, it is 1 h, and in addition, the polymerizationprocess was repeated once.p-AP linkermolecule anchored on the graphene surface. Similar processes havebeen studied by the radical polymerization of acrylate monomers, suchas poly(hydroxyethyl methacrylate), PHEMA, or poly(butyl methacrylate),PBMA, to obtain nanocomposites based on the reaction of the monomerwith nanosilica or by the addition of graphene oxide (GO).52 This reaction is selective and requires the presence of oxygen-containinggroups in order to accomplish the attachment to the acrylate and,therefore, can be a marker of the presence of free −OH groupsin the linked p-AP. The resulting graphene–polymersamples are washed using ethanol to remove the possible physisorbedmonomer and polymer . The final anchoring of the monomer to thelinker molecule covalently bonded on the graphene surface is depictedin x substrates.The process of anchoring the firstmonomer unit to our functionalized graphene surface proceeds as follows.Initially, as the temperature increases, the initiator (AIBN) fragmentsinto primary radicals by the cleavage of weak bonds. These radicalsfind a monomer molecule to react with their double bond and startthe reaction. During polymerization, several monomer molecules areadded from the cleaved double bond, thus forming macroradical chainsattached to this first monomer represented in 33.1p-AP/L-PHEA)and another with a higher polymer coverage (HOPG/p-AP/H-PHEA). Two types of samples were polymerized in situ with HEA, one witha low amount of polymer on the surface . For the HOPG/p-AP sample, this peak iswider and shifted to slightly higher values because of the surfaceroughening and the p-AP molecules. The rougheningas well as the different morphology allows us to confirm the incorporationof p-AP molecules onto the surface. p-AP/L-PHEA) and highest (HOPG/p-AP/H-PHEA) polymer coverage on graphene, respectively.The wide images in p-AP/L-PHEA, a surface coverage close to 20% and a surface densityof about 350 structures/μm2 are observed. A highdensity of features, with heights between 0.3 nm and 4.6 nm is found,a majority of them being of globular morphology with an average heightaround 1.2 nm. These changes confirm that polymerization has takenplace and that it is stable as the samples were measured after beingwashed with water. Finally, for the HOPG/p-AP/H-PHEAsample, the large image in p-AP molecule through the hydroxyl group, and then fromthere the polymer grows. Larger aggregates represent a larger polymerchain. In addition to the domains observed as aggregates in 3.2p-AP-graphite and PHEA-p-AP-graphitearchitecture. p-AP functionalization (HOPG/p-AP), and HOPG samples after the two polymerization processes(HOPG/p-AP/L-PHEA and HOPG/p-AP/H-PHEA).The C 1s core level . We also check that the main sp2 component remainsalmost unchanged, confirming that no damage was induced in the sampleby plasma-assisted processing. After the polymerization, new contributionsappear at 285.2 and 286.0 eV, which are ascribed to C–OH andC=O, respectively, from the hydroxyls and carboxylic groupsfrom PHEA. Finally, relative to the last C 1s spectrum of p-AP/H-PHEA sample reveals the increment of polymer conversionon the graphite surface with the enhancement of the contributionsat 285.2, 286.0, and 286.8 eV, assigned to the oxygen bonds, C–OH,C=O, and O–C=O respectively, from the hydroxylsand carboxylic groups from the PHEA structure.58 This signalenhancement is a direct consequence of the increase in the polymercoverage, in good agreement with the previous morphology seen in The chemical nature of the hybrid functional material has beencharacterized by XPS. Freshly cleaved HOPG is used as a referencesubstrate to study the chemical variations induced in the consecutivestages, the 52 The next spectrumcorresponds to the p-AP covalent functionalized sample,HOPG/p-AP, where, in addition to the adsorbed species,a new component at 532.8 eV appears. We ascribe this new componentto the OH groups of the p-AP molecule, confirmingits presence.60 We also see the increase of adsorbed species,which is easy to explain considering the plasma activation of theHOPG surface and exposure to ambient air before the XPS analysis.This activation enhances the adsorption of molecules as the samplesurface is not completely saturated during the covalent functionalizationexperiment. This intentioned saturation control allows us to maintainthe proper electronic properties of graphene, as we see below . Regardingthe functionalized surface after the polymerization reaction, as expectedfor a PHEA coverage, there are multiple components in the O 1s peak:531.6, 532.8, and 533.7 eV, assigned to the carbonyl (C=O),hydroxyl (OH), and ester (O–C=O) species, respectively.61 Two similar spectra are observedin both L-PHEA and H-PHEA polymerizations, with the only differencein the intensity of each characteristic contribution being largerafter H-PHEA polymerization, as expected. We also observe a smallremaining contribution due to the adsorbed species at 529.9 eV thatmay be a slight surface oxidation due to the existence of steps, defects,grain boundaries or exposed atomic sites after repeated surface washings.For the pristine sample, the spectrumpresents two small componentsthat can be assigned to the environmental adsorbed species as a consequenceof the exfoliation in air.3.3Figures S1 and S2 in the SupportingInformation). The analyses confirm, overall, that the functionalizationprocess takes place similarly on graphene as remarkable differencesin the final morphology of the samples or in the chemical mechanisminvolved are not observed. We conclude that the synthesis of a polymer/graphenehybrid functional material successfully works, which opens the doorto its application in devices.The study from HOPG has allowed us todevelop, characterize and validate the efficiency of a two-stage graphenefunctionalization process based on a linker and functional moleculeattachment on the surface to obtain a graphene–polymer hybridplatform. However, from the large-scale application point of view,the relevant next step consists of the transfer of this protocol toa graphene sample. In this scenario, the substrate morphology andthe structure of functionalized graphene samples have been sequentiallycharacterized by AFM and Raman spectroscopy in every step of the process(see R□) of thelayer by means of a four-point probe analysis at room temperature. R□ variation of the samples, extractedfrom the analysis of the slope of the corresponding I–V curves performed at several points ofthe samples under room conditions . Theinitial pristine graphene layer exhibits an average sheet resistancevalue of 0.55 kΩ after the cleaning procedure that we applyto our samples, 2 h at 340 °C, in a high vacuum ,being a standard result due to the high quality of the material. Thisresistance value increases to 1.05 kΩ on average after the functionalizationwith p-AP, as expected, considering the induced sp3 hybridization of the graphene lattice. As mentioned above,the functionalization density, and thus the hybridization degree,obviously depends on the plasma-assisted processing time, 30 s, asthis parameter determines the precursor dose. The processing timeused is selected after studying its influence on the electrical propertiesand the structure of graphene as a balance between the functionalizationdensity desired and the reduction of conductivity allowed for theintended application . After the polymerization step, the sheetresistance increases, up to 1.35 kΩ, as seen in the last pointof I–V curves measured on each sample. Finally, 64 A similar behavior is observed when p-AP is covalentlybonded to graphene, G/p-AP in red, which does showa small increase in resistivity as the humidity increases, in agreementwith previous reports on this effect as a function of graphene doping65 and band gap opening.66One of the main challenges ofcovalent graphene functionalization is to retain suitable electricalproperties of the graphene layer after processing. In order to studythe changes in the conductivity of graphene throughout the differentstages, we have measured systematically before and after each stagethe sheet resistance  shows that the 2D peak position is around 2682 cm–1 pointing out toward n-doping.68 The water absorption is known to induce p-doping in graphene, aswater molecules behave as electron acceptors69 when directly into contact with the graphenesurface. As the Raman 2D peak of the functionalized samples is inthe same position as that of pristine graphene in Figure S2d in our samples, the water-induced charge transferdecreases the graphene n-doping, increasing its resistivity.However, when the polymer is anchored to the surface,the sensitivityof this graphene-based hybrid platform, in blue, becomes outstanding.This is related to the hydrophilic character of the polymer and itsability to absorb water into its structure.p-AP exhibit a low interactionwith the dynamic humidity change in the environment (red and greenlines). However, a remarkable improvement in the detection performanceof the G/p-AP/L-PHEA material is observed due tothe natural adsorption and release of water molecules on the polymer(blue line). From a quantitative point of view, the resistance variationsor sensitivity, SR, exceed 29% (R□ (94%RH) = 1.57 kΩ, R□ (0%RH) = 1.21 kΩ, from SR in Table S1). Table S1 compiles a performance comparison ofthis work with a number of resistive humidity sensors reported inthe literature70 that confirms the high sensitivityof the hybrid material to the humidity conditions. It is thus demonstratedthat chemical modification by poly-2-hydroxyethyl acrylate on thesurface is a feasible way to improve the sensitivity of graphene tohumidity.Regarding the reproducibility and sensitivity of the materialsas a sensing platform, 4p-AP). This unexploredphysical protocol is clean, reproducible, easily scalable, and cost-effective.The second stage consists of in situ radical polymerization on thefunctionalized graphene surface, leading to a PHEA/graphene hybridmaterial. This strategy is highly versatile and opens the door forthe design of a wide range of graphene–polymer or biopolymerhybrid platforms for biosensing devices. AFM, XPS, Raman, and four-pointprobe sheet resistance analyses confirm that the graphene/p-AP/PHEA architecture is covalently anchored by sp3 hybridization of graphene, retaining suitable electronicproperties after the polymerization process. We have successfullytested the hybrid material as a resistive humidity sensor since thehydrophilic PHEA polymer modifies the hydrophobicity of graphene,finding an outstanding sensitivity to humidity variations, SR = 29%, competing with previous sensors basedon graphene, graphene oxide, reduced graphene oxide, or polymer-functionalizedgraphene. In addition, this proposed graphene–polymer hybridfunctional material leads the way for further biotechnological applicationsthat will be studied in future research projects in order to expandits field of potential applications.In summary,we present a novel route to synthesize a polymer/graphenehybrid functional material that consists of a two-stage process. Inthe first stage, a low-vacuum hydrogen-assisted plasma activationprocess produces a stable and covalent graphene-functionalized platformwith a small linker molecule (


RBD affibody ligands. Molecular docking was performed using HADDOCK software, and three potential ZRBD affibodies, ZRBD-02, ZRBD-04, and ZRBD-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of ZRBD affibodies to RBD was driven by electrostatic interactions. Per-residue free energy decomposition analysis further substantiated that four residues with negative-charge characteristics on helix α1 of the Z domain participated in this process. Binding affinity analysis by microscale thermophoresis showed that ZRBD affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for ZRBD-07 among the three potential ZRBD affibodies. Herein, ZRBD-02 and ZRBD-07 affibodies were selected for chromatographic verifications after being coupled to thiol-activated Sepharose 6 Fast Flow (SepFF) gel. Chromatographic experiments showed that RBD could bind on both ZRBD SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the ZRBD-07 SepFF gel had a higher affinity for RBD. This research provided a new idea for the design of affibody ligands and validated the potential of affibody ligands in the application of RBD purification from complex feedstock.The outbreak of coronavirus disease 2019 (COVID-19) has sparked an urgent demand for advanced diagnosis and vaccination worldwide. The discovery of high-affinity ligands is of great significance for vaccine and diagnostic reagent manufacturing. Targeting the receptor binding domain (RBD) from the spike protein of severe acute respiratory syndrome-coronavirus 2, an interface at the outer surface of helices on the Z domain from protein A was introduced to construct a virtual library for the screening of Z Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), has become a major threat to human health . Within TM) technology, combinatorial search algorithms could search for a tractable number of sequences to satisfy the design criteria from the initial sequence space containing 1050 sequences or more [An important prerequisite of the application of affinity chromatography as well as diagnostic reagents is to design and develop high-affinity ligands targeted to viral particles and vaccines. Wrapp et al. found that single-domain antibodies isolated from a llama immunized with prefusion-stabilized SARS-CoV-1 S protein had cross-reactivity to SARS-CoV-2 and could neutralize SARS-CoV-2 spike pseudotyped viruses . Further or more . In rece or more ,26,27 an or more . In a pr or more , two proStaphylococcal protein A (SpA) [® technology [Among all proteinaceous ligands, affibodies are an important class of protein scaffolds based on the three-helix bundle Z domain derived from  A (SpA) . As a sm A (SpA) . In rece A (SpA) ,33 but a A (SpA) , diagnos A (SpA) . Malm etchnology . Howeverchnology . More imRBD affibody for the screening of high-affinity affibody ligands. Based on different strategies of affibody modeling, several ZRBD affibodies with high affinity for RBD were obtained in a combination of molecular docking and molecular dynamic (MD) simulation. Then, the binding affinity of the affibody to the RBD was validated by isothermal titration calorimetry (ITC) and microscale thermophoresis (MST). Finally, the high-affinity ZRBD affibodies were coupled on Sepharose 6 Fast Flow (SepFF) to evaluate the applicability of affibody chromatography in the purification of RBD.In this study, interfaces at the outer surfaces on helices α1 and α2 of the Z domain  and α2  of the Z domain  and other terms  [RBD binding with RBD, all HADDOCK scores ranged from −74.1 to −137.6, but only three ZRBD affibodies exhibited more favorable HADDOCK scores in RBD binding, namely, ZRBD-02, ZRBD-04, and ZRBD-07. In wild-type ZRBD binding with the RBD, however, the HADDOCK score was only −108.6. This result further indicated that the mutation of ZRBD induced a great change in electrostatic energy in complexes, and the most negative electrostatic energy was observed in the ZRBD-04/RBD complex, as listed in RBD-01), it led to a serious decrease in electrostatic energy in RBD-05 and ZRBD-06) brought about a similarly great decrease in electrostatic energy. However, the dual K4Q/K7D mutation (ZRBD-07) led to more negative electrostatic energy than the single mutation of K4Q (ZRBD-01) and K4G/K7Q (ZRBD-05). On the other hand, ZRBD binding with the RBD also exhibited more negative values of VDW energy in the complexes, and the most negative VDW energy was observed in the ZRBD-02/RBD complex. Therefore, ZRBD-02, ZRBD-04, and ZRBD-07 were further analyzed to determine the structural characteristics of the ZRBD–RBD complexes by MD simulation.The affinity of Ze areas) . The doce areas) . Moreovee areas) . In ZRBDRBDs were examined in a 50 ns MD simulation. The result in RBD (dmin) remained stable at approximately 0.11 nm for the three ZRBD molecules in 50 ns, indicating that RBD and ZRBD maintained effective contact [Rg). The other two sets of trajectories of structural parameters are shown in RBD molecules during MD simulation. As an index of complex compactness, Rg converged rapidly into a range from 2.03 to 2.16 nm in Rg were obtained in RBD binding with ZRBD-04 and ZRBD-07. Both the complexes had slightly extended structures compared with the ZRBD-02/RBD complex. In this study, ZRBD-02 was generated by replacing six solvent-accessible residues at the outer surface of helix α2 of the Z domain to mimic ACE2–RBD binding, as listed in RBD-04 and ZRBD-07. In the latter, only one to two residues were substituted. The results of the MD simulation further demonstrated that the three potential ZRBDs obtained from molecular docking formed stable complexes with the RBD.The structural fluctuation and stability of the complexes of the RBD and three potential Z contact . Figure EC) and Lennard–Jones (L-J) energies (EL-J) between the RBD and three ZRBD molecules. The other two sets of time trajectories of EC and EL-J are shown in EC just fluctuates around −14.1 MJ/mol during the MD simulation, as shown in EL-J fluctuated at approximately 1.30 MJ/mol. Among the three complexes, negative values of EC signified that the binding was driven by electrostatic interactions.RBD by the MM-PBSA method [Gbind) of RBD complexes with ZRBD-02, ZRBD-04, and ZRBD-07 were determined to be −27.4, −128.6, and −405.6 kJ/mol, respectively, as listed in RBD-07 had the highest binding affinity to the RBD among the three potential ZRBD ligands . In this process, electrostatic interactions (ΔGelec) dominated the formation of ZRBD/RBD complexes. Moreover, VDW interactions (ΔGVDW) also favored ZRBD binding to the RBD whereas the polar solvation free energy (ΔGPB) disfavored binding. Herein, per-residue free energy decomposition was performed to further analyze the contribution of each residue pair to the binding free energy of the ZRBD-04/RBD and ZRBD-07/RBD complexes. The result is shown in RBD-04/RBD complex, four key residues, E8, D10, E15, and D18, of ZRBD were highlighted as shown in RBD-04/RBD complex, moreover, an additional negatively charged residue (D7) was found in the ZRBD-07/RBD complex, as shown in RBD binding to the RBD.In this study, the g_mmpbsa tool of GROMACS was used to calculate the binding free energy of the RBD and ZA method . The binRBD affibody were measured with circular dichroism (CD) and fluorescence (FL) spectrometers. As shown in RBD-07 affibody. The FL emission spectrum was applied to analyze the tertiary structure of ZRBD in solution. The results in RBD had a maximum emission intensity at approximately 340 nm, indicating a stable tertiary structure of the affibody in solution. However, a slight red shift of the FL emission peak was observed in ZRBD-04, demonstrating that the chromogenic Tyr residue in the native state was exposed to a slightly more hydrophilic environment and led to a slight change in tertiary structure [RBDs maintained their molecular structure and that ZRBD-02 and ZRBD-07 were more stable in solution.In this study, the spectral characteristics of the Ztructure . SpectraRBD and RBD led to a marked variation in normalized fluorescence, and all the curves had a reverse sigmoidal shape in the thermophoresis signal with an increase in affibody concentration until the binding approached saturation. By fitting the normalized fluorescence signal and affibody concentration, the dissociation constants of RBD binding were determined to be 133.4 nmol/L for ZRBD-02, 377.3 nmol/L for ZRBD-04, and 36.3 nmol/L for ZRBD-07. These values of the dissociation constant were superior to those in the peptide–RBD binding (80–970 nmol/L) [RBD-02 and ZRBD-07 had higher affinity for the RBD than ZRBD-04 and even the binding affinity of ZRBD-07 was an order of magnitude higher than that of ZRBD-04. In this study, the raw ITC data in RBD-02 and ZRBD-07 exhibited typical exothermic characteristics. This evidence was consistent with previous results of MD simulations.In this study, the binding affinity of affibody to the RBD was evaluated with microscale thermophoresis (MST). The results in  nmol/L) . The resRBD-02 and ZRBD-07 with the C-terminal CK tag were coupled to thiol-activated SepFF gel to synthesize two affinity adsorbents (ZRBD-02 SepFF and ZRBD-07 SepFF) for chromatographic experiments. The ligand densities were determined to be 6.5 mg/mL gel for ZRBD-02 SepFF and 7.3 mg/mL gel for ZRBD-07 SepFF. The chromatographic results are shown in ® Epoxy 85 resin [RBD-02 SepFF and ZRBD-07 SepFF gels. The SDS–PAGE results further showed that lane E3 of RBD-07 SepFF gel had a higher binding amount of RBD at a similar ligand density. Moreover, low-molecular-weight components in lane E3 of RBD-02 via disulfide linkage because the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum  were synthesized by Ziyu Biotech Co., Ltd. . Recombinant His-tagged SARS-CoV-2 Spike RBD (amino acids Arg319-Phe541-His6) and hACE2 expressed in SF9 insect cells were obtained from Nankai University [SepFF gel was purchased from Cytiva . Ziversity . HPLC-grRBD affibodies in the library were built manually via homology modeling. The sequence of the recombinant Z domain from SpA (PDB code: 2SPZ) was retrieved in FASTA format from the Protein Data Bank for the preparation of amino acid sequences of the ZRBD affibody in this study. The modeling of the affibodies’ structures was performed by SWISS-MODEL . Once designed, either ZRBD affibodies or ZRBD affibodies with a C-terminal CK tag (ZRBD-Cys-Lys) were synthesized.In this study, solvent-accessible residues at the outer surfaces of helices α1 and α2 of the Z domain . Prior to docking, the RBD and ZRBD affibodies were prepared by PyMOL software to remove other protein chains, water molecules, and ions from the PDB file and insert missing H-atoms. After two prepared PDB files were uploaded, the amino acid 470–510 region was selected as the RBD docking region and the amino acid 1–19 region was selected as the affinity ligand docking region according to the binding mechanism of RBD to ACE2 [Eelec, Evdw, EAIR, and Edesolv).In this research, a highly fuzzy-driven docking method named HADDOCK (high ambiguity driven protein–protein docking) was used to create the model of the RBD–affibody complex. HADDOCK permits the utilization of biochemical or biophysical interaction data, such as chemical shift perturbation data or mutation data from NMR titration experiments, and information about interaction residues is introduced as ambiguous interaction constraints (AIRS) to drive the docking process . Crystal to ACE2 ,49. The  to ACE2 . The finRBD affibodies. In simulation system 1, a regular dodecahedral box was generated to solvate the RBD and ZRBD-02, which contains 10,536 water molecules and 1 chloride ion . In simulation system 2, a regular dodecahedral box was generated to solvate the RBD and ZRBD-04, which contains 10,441 water molecules and 1 chloride ion . In simulation system 3, a regular dodecahedral box was generated to solvate the RBD and ZRBD-07, which contains 10,463 water molecules and 1 chloride ion .The structures of SARS-CoV-2 RBD and affibody complexes used for model construction were built from HADDOCK. The CHARMM36 force field was used and the TIP3P model was used for water molecules. In this study, three simulation systems were constructed using models including RBD complexes with different Zhttp://www.gromacs.org/, accessed on 10 October 2022). The temperature was controlled at 300 K with a time constant of 0.1 ps using the velocity-rescale  method [x, y, and z directions. After the energy was minimized using the steepest descent method, a 50 ns MD simulation was performed as described. Every MD simulation was repeated to obtain three independent simulation trajectories for sufficient sampling and analysis.MD simulations in the NVT ensemble were performed using GROMACS version 2018.1-gpu  method . The cutRg as well as dmin were calculated to quantitatively evaluate the stability of protein constructs. RMSD was calculated by the rms program of GROMACS software. The larger the RMSD, the larger the structural change during docking. Rg was calculated by the gyrate program of GROMACS software. The smaller the Rg, the more compact the molecular structure. The g_mmpbsa tool from GROMACS was applied in the implementation of the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) approach for end-state free energy calculations from MD trajectories and binding free energy decomposition [RBD affibodies.In this study, RMSD and position . TherefoCD spectra of free affibodies were determined with a JASCO J-810 spectro-polarimeter from JASCO Inc.  with a 1 mm path cuvette at room temperature. In this study, 20 mmol/L PB buffer (pH 7.5) was used as the sample buffer unless otherwise specified. Prior to the analysis, the affibodies were diluted to 0.2 mg/mL with the sample buffer. The data were collected at 0.1 nm intervals at a scanning speed of 100 nm/min in a wavelength range of 190–240 nm. In the measurement, the sample buffer was used as a reference. After three consecutive wavelength scans were taken for each sample, the spectra were averaged and then corrected by subtracting the spectrum of a reference.FL emission spectra of free affibodies were analyzed with a PerkinElmer LS-55 fluorescence spectrophotometer in the range of 280–400 nm (emission slit width of 7.5 nm) at the excitation wavelength of 284 nm (excitation slit width of 12.0 nm). All the samples were measured in a quartz colorimetric dish with a 10 mm path, and the scanning speed was 300 nm/min. The spectral analysis was repeated three times for each sample. The fluorescence background was determined using the sample buffer, and the emission spectra of the samples were corrected by subtracting the FL background.Heat variation in the affibody and RBD binding was measured with an Affinity ITC from TA Instrument Company  at 25 °C and a stirring speed of 200 rpm. Prior to the ITC experiment, affibody samples were dissolved in the sample buffer with a final concentration of 250 μmol/L, and the RBD stock solution was diluted to 22 μmol/L with the sample buffer. After the samples were degassed at 25 °C for 10 min, the degassed RBD solution (500 μL) was slowly transferred into the titration cell, and the affibody solution was inhaled into the ITC syringe. In the titration, the affibody solution was injected into 21 injections of 2 μL each at intervals of 300 s. As a reference, the sample buffer was injected into the RBD solution to determine the heat of dilution. All experiments were repeated in triplicate.RBD binding to RBD was performed with a Monolith NT.115 Microscale Thermophoresis from Nano Temper Technologies GmbH . Prior to the measurement, the dye was labeled on the RBD via a C-terminal His6 tag using a Monolith His-Tag Labeling Kit. The concentration of the labeled RBD was 0.05 μmol/L, and the concentrations of the ZRBD affibodies were in a gradient dilution from 2.5 μmol/L in 16 PCR tubes. After the labeled RBD was transferred into PCR tubes, the reaction was incubated and then loaded into Monolith NT.115 capillaries. MST was carried out in MST buffer  at 20% red channel LED and 20% MST power.MST of ZRBD affibodies with a C-terminal CK tag were coupled onto SepFF via the thiol immobilization technique. It included the synthesis of a thiol-modified gel (presented in 3 (3 mL). After 0.17 g/L DTT in 1 mmol/L EDTA solution (3 mL) was added, the mixture was reacted at 25 °C for 30 min. The gel was rinsed with 0.2 mol/L NaHCO3 and 1 mmol/L EDTA solution, and dried in a G3 sintered glass funnel. Mercaptopyridine (MPy) was coupled to the gel as described by Ferraz et al. [v/v) containing 0.06 mmol/L EDTA and 20 mmol/L NaHCO3 (50 mL), the drained gels (3.0 g) were mixed with an acetone–water mixture  and 0.3 mol/L DPDS (10 mL). The slurry was reacted at 25 °C for 1 h. The product was collected and washed with an acetone–water mixture (3/2 v/v) and 1 mmol/L EDTA and denoted SepFF-MPy.Zz et al. . After tRBD affibodies, ZRBD-02 and ZRBD-07, onto SepFF-M, 20 mmol/L PB buffer (pH 7.5) was used as the coupling buffer. After 1.0 g SepFF-MPy was washed with the coupling buffer, the gels were mixed with 10 mg ZRBD affibody in coupling buffer (5 mL), and the slurry was reacted at 25 °C for 1.5 h. Then, cysteine was added, and residual MPy on the gels was replaced by cysteine after reacting continuously for 30 min. In this study, two final products were denoted ZRBD-02 SepFF and ZRBD-07 SepFF for chromatographic experiments.In the coupling of ZRBD SepFF gel (1 mL) connected to an AKTA Purifier 10 chromatography system . After the column was equilibrated with adsorption buffer , the RBD sample was injected, and then the column was washed with 6 column volumes (CVs) of adsorption buffer to remove unbound components. Finally, the column was eluted with 0.5 mol/L NaCl in adsorption buffer, 0.1 mol/L Gly-HCl buffer (pH 3.0), and 0.1 mol/L NaOH. The collected eluted fraction at pH 3.0 was neutralized with 1.0 mol/L Tris-HCl buffer (pH 8.0) at a proportion of 5% (v/v). Cleaning in place (CIP) was carried out with 0.5 mol/L NaOH. The purity of the RBD in the flow-through and elution fractions was analyzed by SDS–PAGE and the leakage of affibody ligand was determined with a MALDI-TOF mass spectrometer.Chromatographic experiments were performed at 0.2 mL/min (1.0 cm/min) using a Tricorn 5/50 column packed with Z® LP-C18 column from Welch Materials Inc.  at 220 nm using 0.2% TFA in water as mobile phase A and 0.2% TFA in acetonitrile as mobile phase B. In the analysis, a linear gradient of mobile phase B from 20% to 70% in 25 min was applied. Purity analysis was performed with 15% non-reducing SDS–PAGE gel. The electrophoresis was run at a constant voltage of 120 V until the dye front reached ~1 cm from the bottom of the gel. The gel was stained with Coomassie brilliant blue R-250. After decolorization, the gel was analyzed by imaging and grayscale calculation using the gel analysis system from Shenhua Technology to determine the purity of the RBD. The elution components were analyzed with a Bruker Autoflex III MALDI-TOF mass spectrometer . The spectrum from 5 kDa to 8 kDa was collected in positive ion mode.The change in affibody content during ligand coupling was measured with a Shimadzu Essentia LC-16 chromatographic system  using an UltimateRBD candidates were generated and three ZRBD affibodies with lower HADDOCK scores than the ACE2/RBD complex, ZRBD-02, ZRBD-04, and ZRBD-07, were obtained. MD simulation verified that the three ZRBD affibodies formed stable complexes with the RBD and this process was driven by electrostatic interactions. Based on the per-residue free energy decomposition, four residues with negative-charge characteristics were further highlighted for the formation of the complexes. CD and FL emission spectra showed that the ZRBD affibodies maintained their molecular structure in solution and this phenomenon was more pronounced for ZRBD-02 and ZRBD-07. Although three ZRBD affibodies had a binding affinity to RBD, higher binding affinities were obtained in the binding of the RBD with ZRBD-07 (Kd = 36.3 nmol/L) and ZRBD-02 (Kd = 133.4 nmol/L). Herein ZRBD-02 and ZRBD-07 affibodies were selected for chromatographic verifications. Chromatographic results showed that both ZRBD-02 SepFF and ZRBD-07 SepFF gels could bind the RBD effectively and the latter had a higher binding amount of RBD. The bound RBD was eluted by 0.1 mol/L NaOH, indicating strong binding of the RBD on ZRBD SepFF gels. This research provided a new idea for the design of affibody ligands, and the results demonstrated that affibodies derived from the Z domain of SpA were a potential protein scaffold for the design of high-affinity ligands in the application of RBD purification from complex feedstock.In this study, the interface at the outer surface of helices α1 and α2 on the Z domain from SpA was introduced for ligand design. Based on different strategies for affibody modeling, eight Z


Chlorella vulgaris to remove arsenic from aqueous solutions. A series of studies was conducted to determine the optimal conditions for biological arsenic elimination, including biomass amount, incubation time, initial arsenic level, and pH values. At 76 min, pH 6, 50 mgL−1 metal concentration, and 1 gL−1 bio-adsorbent dosage, the maximum removal of arsenic from an aqueous solution was 93%. The uptake of As (III) ions by C. vulgaris reached an equilibrium at 76 min of bio-adsorption. The maximum adsorptive rate of arsenic (III) by C. vulgaris was 55 mg/gm. The Langmuir, Freundlich, and Dubinin–Radushkevich equations were used to fit the experimental data. The best theoretical isotherm of Langmuir, Freundlich, or/and Dubinin–Radushkevich for arsenic bio-adsorption by Chlorella vulgaris was determined. To choose the best theoretical isotherm, the coefficient of correlation was used. The data on absorption appeared to be linearly consistent with the Langmuir , Freundlich , and Dubinin–Radushkevich  isotherms. The Langmuir and Dubinin–Radushkevich isotherms were both good two-parameter isotherms. In general, Langmuir was demonstrated to be the most accurate model for As (III) bio-adsorption on the bio-adsorbent. Maximum bio-adsorption values and a good correlation coefficient were observed for the first-order kinetic model, indicating that it was the best fitting model and significant in describing the arsenic (III) adsorption process. SEM micrographs of treated and untreated algal cells revealed that ions adsorbed on the algal cell’s surface. A Fourier-transform infrared spectrophotometer (FTIR) was used to analyze the functional groups in algal cells, such as the carboxyl group, hydroxyl, amines, and amides, which aided in the bio-adsorption process. Thus, C. vulgaris has great potential and can be found in eco-friendly biomaterials capable of adsorbing arsenic contaminants from water sources.This study examined the ability of the green microalgae  Heavy metals are found randomly and naturally in the layers of the Earth and water sources, but they negatively affect human life if they exceed internationally permissible concentrations ,2. It isArsenic is a naturally occurring element in the Earth’s crust and is widely distributed throughout the environment, including in water, air, and soil. It is exceedingly poisonous in its inorganic form and is one of ten compounds classified by the World Health Organization as a significant public health concern . The curPhormidium tenue and C. vulgaris, as bio-sorbent materials to remove them from aqueous solutions [Heavy metals are not biodegradable because they do not decompose or deteriorate when they penetrate the cells of living organisms ,15,16. Tolutions .C. vulgaris has lately gained popularity for treating heavy metal pollution in aquatic environments [C. vulgaris, which was employed in this study to observe its ability to adsorb arsenic from an aqueous external environment.Algae create more biomass than other microbes and, for the most part, do not produce hazardous chemicals . C. vulgronments ,29. The 2 was made at a concentration of 1000 mgL−1 as the source of arsenic (III). After that, the solutions were diluted to the desired concentration and tested [The stock solution of NaAsOd tested . All cheChlorella vulgaris (SAG 211-11b) was the name of the microalgal strain employed in this study. This strain was purchased from Goettingen University’s Culture Collection of Algae and re-cultured in 10 mL of Chlorella vulgaris-appropriate Fog’s Medium.These steps were used to prepare the microalgal biomass. First, the algal biomass was deactivated in an autoclave at 121 °C for 10 min. Second, the deactivated biomass was collected by filtering cultured media using cellulose nitrate Whatman membranes with pore sizes of 45 m and washed with deionized water. Third, the biomass was sun-dried for three days before being oven-dried at 55 °C for 24 h and pulverized in a stone mortar and pestle to achieve a uniform particle size. For future usage, forty sieved materials were stored in an airtight container .C. vulgaris were determined in batch mode at 30 ± 1 °C using several parameters such as pH , biomass dose , contact interval , and initial concentration . Experiments were carried out in three copies in a 150 rpm orbital incubator using 250 mL conical flasks containing 100 mL of arsenic solution. After centrifugation incubation at 4000 rpm for 10 min, the microalgal biomass was separated. The supernatant was filtered via 0.45-micron Millipore filters, and the filtering was tested for residual arsenic using an atomic absorption spectrometer set at 283.3 nm .The optimal conditions for arsenic adsorption by The chemical constituents of the algal adsorbent  were analyzed using an elemental analyzer .−1 wave number range.The functional groups of the algal adsorbent and arsenic (III)-loaded algal adsorbent were further investigated using a Fourier-transform infrared (FTIR) spectrophotometer  in the 400–4000 cmThe untreated and arsenic-treated adsorbents were collected and prepared for the SEM  examination .C. vulgaris) was utilized in the adsorption isotherm analysis under conditions such as an adsorbent dosage of 1 g, varied initial arsenic (III) concentrations (10 to 300 mgL−1), and agitation at 150 rpm and 25 °C for 8 h to ensure that the adsorption system attained an equilibrium, with the initial solution pH adjusted to 6.0.The optimum concentration of chemical reagents (1 M NaCl for −1) of the As (III) was calculated following Equation (1):0 (mgL−1) represents the initial As (III) concentration. Ce (mgL−1) represents the (III) concentration at the equilibrium.The amount of equilibrium adsorption (qe mggmax (mgg−1) represents the maximum adsorption capacity, qe (mgg−1) represents the equilibrium-adsorbed amount of As (III), and Ce (mgL−1) denotes the solute’s equilibrium concentration. The Langmuir constant for adsorption energy is represented by KL, whereas KF is the magnitude of bio-adsorption capacity; n is the bio-adsorption intensity, KD–R and β are D–R constants, and E is energy for the uptake of arsenic on the bio-adsorbate surface.The Langmuir and Freundlich models  in Equat−1 As (III), 1 gL−1 adsorbent, pH 6, and agitation at a speed of 150 rpm and a temperature of 25 °C, with time intervals ranging from 5 min to 8 h, depending on the appropriate equilibrium time. The adsorption kinetic data on the removal of As (III) were assessed via pseudo-first- and second-order models. Equation (8) expresses the pseudo-first-order kinetic model.The bio-adsorption kinetics were investigated in 50 mL of solution under conditions such as 30 mgLe and qt represent the equilibrium adsorption capacity (mgg−1) and the adsorption capacity at t (min), respectively, and k1 and k2 are the rate constants for the pseudo-first-order and pseudo-second-order models, respectively. All the experiments were duplicated and the averages were used for the analysis.Equation (9) shows the pseudo-second-order kinetic model:Chlorella vulgaris was investigated by varying the temperature from 10 to 50 °C at optimum conditions and evaluating the temperature effect using the following equations:The uptake of arsenate ions by −1 arsenic, 1 g bio-adsorbent, and a 120 min incubation was determined using a pH range of 1–8. The bio-adsorption rate was limited at an initial pH of 3.0 and reached a maximum at an optimal pH of 6.0, with a bio-adsorption rate of 58 mgg−1; the removal percentage was 63%. According to Abdel-Raouf and colleagues, determining the optimal pH has a significant impact on regulating the metal aqueous and functional groups of adsorbent surfaces, thereby influencing heavy metal adsorption in an aqueous environment [+ hydrogen ions, the adsorption capacity diminishes when the pH falls below 3. In this instance, the rate of arsenic ions in the solution was lower than the concentration of H+ ions, increasing the possibility of competition with them for surface binding sites of adsorption due to repulsive forces between metal ions and proteins [The bio-adsorption capability was highly dependent on pH, as shown in ironment . Bio-adsironment . Due to proteins . The absC. vulgaris at various contact times . After 76 min of bio-adsorption, C. vulgaris’ uptake of As (III) ions reached an equilibrium. The time it takes for heavy metals in a solution to reach a constant value is known as the adsorption equilibrium time. The maximum bio-adsorption rate for As (III) uptake was 55 mgg−1 and the removal percentage was 64%. According to a similar study, a rapid absorption rate was followed by a slower rate and, eventually, an equilibrium [ilibrium . The rat−1 led to a rise in the As (III) ion removal percentage, from 8 to 100%. The removal percentage remained unchanged after the concentration reached 1 gL−1. In contrast, increasing the bio-adsorbent dosage between 1 and 5 mgg−1 reduced the bio-adsorption rate from 55 to 20 mgg−1. Consequently, the optimal bio-adsorbent dose was found to be 1 gL−1, with the highest rate of arsenic bio-adsorption by C. vulgaris being 55 mgg−1 and removal percentage of 93%. It should be noted that the adsorption rate decreased as the adsorbent dose increased due to the initial metal ion concentration being insufficient to match all the active sites on the adsorbent’s surface. This was an indicator of the attraction between the functional groups present on the adsorbent’s surface and the arsenic particles [The results depicted in articles ,39.C. vulgaris at different concentrations of arsenic ions. The bio-adsorption capacity of As (III) grew proportionally with the initial concentration, registering a 50 mgL−1 bio-adsorption capacity with a 55 mgg−1 removal percentage of 100%. The minimum and maximum As (III) bio-adsorptive capacities were 24 mgg−1 and 55 mgg−1, respectively, with As (III) concentrations of 25 and 50 mgL−1. An increase in the initial As (III) concentration may have resulted in inconsistent metal ions being in contact with the algal absorbent surface, which could have led to mass transfer resistance between the aqueous and absorbent phases. Nonetheless, increasing the As (III) ion concentration from 50 to 200 mgL−1 did not increase the rate of uptake capacity, with the capacity remaining constant at 55 mgg−1. This indicated that near-constant saturation was achieved and that there were limited binding sites available for adsorption [We evaluated the highest concentration of arsenic ions that could be adsorbed by the algal absorbent at a 1 g bio-adsorbent dose with pH of 6.0 and a contact time of 76 min. sorption .The percentages of the most critical element on the algal adsorbent surface  were oxy−1, indicating the existence of O-H and N-H [−1 correlated with the C-H stretching vibrations in cellulose and lignin [−1 were also linked to C=O asymmetric stretching. More significant shifting revealed a tight connection between the metal ions (As (III)) and the algal surface. Plazinski et al. [−1 was due to -SO3 with aliphatic amine stretching (C-N). Compared with unmodified C. vulgaris, the strength of the O-H, C-H, and C=O stretching bands for modified C. vulgaris increased, indicating the increase in these functional groups. The FTIR graph showed that the C. vulgaris surface was occupied by the hydroxyl, carboxylate, amino, and amide groups, which had a remarkable role during the adsorption process [FTIR spectroscopy was used to identify several functional groups present on the surface of the algae. The FTIR spectra of the algal adsorbent revealed several types of vibrational frequencies due to distinct functional groups. The FTIR graphs of the original and chemically transformed algal samples are shown in  and N-H . Accordi and N-H , a prolod lignin . Peaks ni et al.  reported process .The mechanisms of adsorption can be generally categorized as physisorption and chemisorption. The physisorption mechanism involves adsorption between the surface and adsorbate, which includes a range of factors such as diffusion, van der Waals interactions, hydrogen bonding, electrostatic interactions, and exothermal phenomena . On the C. vulgaris. Langmuir, Freundlich, and Dubinin–Radushkevich models were used to describe the equilibrium relationships. 2; As = 0.9894) suited the Langmuir isotherm model better for C. vulgaris than the Freundlich and Dubinin–Radushkevich isotherm models . The Langmuir maximum bio-adsorption capacity (qmax) was 45 mgg−1 whilst in the case of the Dubinin–Radushkevich model, it was 8.7 mgg−1. The As (III) adsorption behavior approximated the Langmuir isotherm better , implying that the nature of adsorption was homogeneous via monolayer adsorption [C. vulgaris could successfully remove As (III) from aqueous solutions.The bio-adsorption isotherm at a given condition represents the equilibrium relationship between the adsorbate concentration in an aqueous solution and that on the adsorbent’s surface of microalgae sorption ,49,50. TC. vulgaris. The values of the constant kinetic parameters for As (III) bio-adsorption are shown in 2 for As (III) calculated from the pseudo-first-order model was significantly higher (0.9801) than the values calculated from the pseudo-second-order kinetic model for C. vulgaris. The observed qe values (3.130 mgg−1) were confirmed by the computed qe values for As (III), derived using the pseudo-second-order model with the adsorbent [2 = 0.9801 for an initial concentration of 50 mgL−1). This indicated that the bio-adsorption of arsenic ions was best through pseudo-first-order kinetics. The slope and intercept of the plot in e and k2.Experiment data were evaluated with pseudo-first-order and pseudo-second-order models to investigate the As (III) bio-adsorption kinetics on dsorbent . Figure −1, ΔS  =  104.320 kJ mol−1 K−1, and ΔG  =  −1.76 to −4.98 kJ mol−1 K−1. Endothermic adsorption was indicated by the positive value of ΔH, whilst the negative value of ΔG implied spontaneous adsorption. The low activation energies of arsenate bio-adsorption on C. vulgaris implied that the sorption of arsenic on the algae could involve both physical and activated processes. However, the positive heat of adsorption of the bio-sorbent indicated that endothermic adsorption occurred [C. vulgaris, indicating an endothermic process. Moreover, all free energy values at various temperatures were negative, indicating that arsenic adsorption on the adsorbents evaluated occurred spontaneously.The temperature parameters during the sorption process are of immense significance. An increase in temperature generally accelerates the rate of chemical reactions. As part of the investigation, the Van’ t Hoff equation and other relations were used to calculate the activation energy of arsenic sorption. occurred . Based oC. Vulgaris (SAG 211-11b). It demonstrates that the qmax values for various adsorbents vary significantly. In this study, C. vulgaris (SAG 211-11b) (20.9 mgg−1 at pH 6) had a potential adsorption efficiency for arsenic (III) from aqueous solutions when compared with Chlamydomonas sp. (53.8 mgg−1 at pH 4), C. vulgaris (13 mgg−1 at pH 6), C. vulgaris modified with NaCl (20.9 mgg−1 at pH 6), and Spirulina platensis modified with ZnCl2 NaCl (24.8 mgg−1 at pH 6). The results in C. vulgaris (SAG 211-11b) as a bio-adsorbent was the best in the bio-adsorption of arsenic (III) from aqueous environments.C. vulgaris to bio-adsorb As (III) simultaneously. To achieve the bio-adsorption of As (III) ions from aqueous solutions, C. vulgaris was employed. The findings indicated that the adsorption rate and removal percentage of As (III) ions were subject to the pH, initial metal concentrations, biomass dosage, and contact time as well as a rougher surface structure and larger specific surface area of bio-adsorbents. The optimal adsorptive ability of As (III) was found to be 55 mgg−1 with a 100% removal percentage. Among the models used, Langmuir was found to be the most precise model for As (III) bio-adsorption on the bio-adsorbent at an uptake of 45 mgg−1 compared with the Freundlich and Dubinin–Radushkevich isotherm models. The research concluded that the bio-adsorption process was endothermic and spontaneous, as per the thermodynamic studies. Consequently, the study proposed an efficient and environmentally friendly solution to simultaneously eliminate As (III) by C. vulgaris (SAG 211-11b).The study aimed to investigate the ability of green algae


Attacins are one of the relatively large AMP families, which show anti-Gram-negative activity in vitro.Upon infection, insect hosts simultaneously express a cocktail of antimicrobial peptides (AMPs) which can impede pathogen colonization and increase host fitness. It has been proposed that such a cocktail might be adaptive if the effects of co-expressed AMPs are greater than the sum of individual activities. This could potentially prevent the evolution of bacterial resistance. However, Tenebrio molitor: (TmAttacin1a (TmAtt1a), TmAttacin1b (TmAtt1b), and TmAttacin2 (TmAtt2) both individually and in combination. We then infected T. molitor with the Gram negative entomopathogen Pseudomonas entomophila.Here, we used RNA interference (RNAi) to silence three members of the Attacin family genes in the mealworm beetle, TmAttacin1b, TmAttacin2 and the knockdown of all three Attacins together. Triple knockdown, rather than individual or double knockdowns of AMPs, changes the temporal dynamics of their efficiency in controlling the colonization of P. entomophila in the insect body.We found that survival of the beetles was only affected by the knockdown of P. entomophila load early in the infection process, resulting in differences in host survival. Our results highlight the importance of studying AMP-interactions in vivo.More precisely, AMP gene expression influences  Priorsolation –8. Insecsolation –11. In asolation , 12.in vivo AMP activities of single AMPs or AMPs in combination. Zanchi et al. . In another study using CRISPR/Cas9-Induced loss-of-function, Hanson et al.   (T. molitor) (Escherichia coli)  by a recombinant Attacin from H. illucens  –18, Lepi exigua) , and Colmolitor)  have beemolitor) , 24. Attia coli) , 19, 22.orsitans , and alsillucens . HoweverT. molitor as a model host to gain insight into the host-pathogen interaction upon bacterial infection and to uncover the importance combinations of Attacins. Previous studies have highlighted that antimicrobial activities of T. molitor AMPs in vitro do not reflect activities in vivo. There are several possible reasons for this such as synergisms among AMPs and local pH and salt concentration  pathway in adult and larval D. melanogaster both systemically and locally in the gut following digestion. This results in the production of AMPs  influences host survival and bacterial load, taking advantage of RNAi to provide single-, double-, and triple-knockdown of the Attacin family genes.We here knockdown members of one AMP family 22.1ad libitum and kept in the dark at 25 ± 3°C. Larvae were supplemented with a piece of fresh apple every two days as a source of moisture and nutrition. Every 48 h pupae were collected and sexed using a dissecting microscope (ZEISS stereo microscope STEMI 305). Previous studies have shown sexual dimorphism in immune function in T. molitor. Therefore, to reduce variation in survival and/or immune traits’ expression due to sex, only females were retained for experiments. These females were kept under the controlled conditions from eclosion onwards . A group of 500 larvae were maintained in plastic containers (20 cm × 20 cm × 9 cm). They were fed wheat bran  onwards . All exp2.2P. entomophila infection influences the expression levels of three TmAttacin genes, T. molitor females were exposed to either PBS or P. entomophila in PBS and expression changes in the Attacin genes confirmed via qRT-PCR. Following this confirmation, single- , double- (TmAtt1a-Att2 and TmAtt1b-Att2), and triple-knockdowns (TmAtt1a-Att1b-Att2) were performed using RNAi. Double-stranded enhanced green fluorescent protein (dsEGFP) was injected as a negative control for the nonspecific effects of dsRNA. In each combination or single knockdown (KD) experiment, we needed to determine the time point at which the depletion in transcripts of our gene (s) of interest compared to dsEGFP was significant in T. molitor females. Therefore, efficiency of RNAi target gene suppression was evaluated in adult females collected from dsRNA- and dsEGFP-treated beetles. Based on these data, we selected the time points for P. entomophila infection. No infections were carried out before wounds from the knockdown injection had sufficiently healed. The immune tissues  were dissected from infected beetles of all treatments for the first three days following infection and the survival of a second set of beetles was monitored daily for 35 days in parallel. A previous study on S. aureus-infected T. molitor females suggests that this time window captures most of the mortality caused by the infection without being confounded by senescence (To examine whether EGFP (negative control) or gene (s) of interest-dsRNA  groups. Each of these groups were divided into two subgroups, injected with PBS (control) or P. entomophila  or one of the three dsRNA-treated groups, dsEGFP and dsRNA (target gene (s) following P. entomophila infection  were recovered from dsRNA-treated beetles only, namely ds2.3P. entomophila L48 (provided by Alexandro Rodríguez-Rojas) was cultured from 50% glycerol frozen stock at 28 ± 2°C in ten mL of Luria-Bertani (LB) broth overnight. 100 μL of overnight culture was harvested and reincubated in ten mL LB broth for a further 2 h under the same conditions. Cultures were interrupted when an optical density of 0.5 at 600 nm (OD600) was reached, measured by spectrophotometer . The subcultured bacteria were concentrated by centrifugation and the cell pellet was washed three times with phosphate buffered saline (PBS) and then was resuspended. Based on the OD600, the culture was adjusted to 1 × 104 cells/µL.4 cells/µL of P. entomophila. Based on preliminary experiments, this infection concentration was likely to yield progressive mortality over time  and measured changes in the expression patterns in our genes of interest using relative quantitative PCR (qRT-PCR). Four individual beetles were pooled together and homogenized with a pestle in liquid nitrogen before being transferred to 2 mL microcentrifuge tubes . Total mRNA was extracted using the Quick-RNA Tissue/Insect Microprep Kit (ZYMO Research Europe GmbH), following the manufacturer’s instructions. The resulting mRNA was stored at -80°C until being used for qRT-PCR. qRT-PCR was performed using Power SYBR™ Green RNA-to-CT™ 1-Step Kit (Applied Biosystems TM) with extracted mRNA and specific primers as listed in T. molitor 60S ribosomal protein L27a (TmL27a) and averaged for each gene. The mean Ct of the AMP gene of interest was normalized using the mean Ct of TmL27a by calculating the mean Ct TmL27a - mean Ct of the gene of interest (delta Ct). Gene expression levels were expressed as 2^ (delta-delta Ct)  .2.5TmAttacin1a (GenBank accession No. MF754109), TmAttacin1b (MF754110), TmAttacin2 (MF754108)  as per the manufacturer’s recommendations. Next, the synthesized dsRNA was washed, and the RNA pellet was resuspended in nuclease-free water and kept at -20°C until further use.To silence target gene expression by RNAi, total RNA was extracted  from the late instar larvae showing the highest enrichment of F754108)  , adult females were injected with 1000 ± 100 ng of dsRNA (500 μg/μL in 2 μL), for double-knockdowns 2000 ± 100 ng (1000 μg/μL in 2 μL) (TmAtt1a-Att2 and TmAtt1b-Att2), and for triple-knockdown (TmAtt1a-Att1b-Att2) 3000 ± 100 ng of dsRNA in 6 µl total volume of nuclease-free water . The same concentration of dsEGFP was injected for control insects, giving three control groups for single-dose, double-dose, and triple-dose controls, respectively.To address the individual and combined functional importance of To confirm RNAi efficiency of target genes, total RNA was extracted  for single- (at first week and 14-day), double- , and triple-knockdown (4- and 5-day) at different time points post-exposure and then qRT-PCR carried out as described in section 2.4.T. molitor females exposed to bacteria after silencing the target genes, there were six treatments in each set  namely, full control - PBS, full control - P. entomophila, dsEGFP - PBS, dsEGFP - P. entomophila, dsRNA - PBS, dsRNA - P. entomophila. Survival assays were replicated twice in a separate set of beetles and bacterial cultures.Next, to evaluate the viability of 2.7P. entomophila successfully survive in the T. molitor tissues or are killed by the host immune system, the immune tissues of TmAttacin knockdown and dsEGFP beetles were dissected on days 1, 2, and 3 post-infections. Sampling was stopped at this time point due to a high number of dead beetles, which led to a reduced sample size. We collected haemolymph, fat body, gut, and Malpighian tubules of each T. molitor female before pooling and homogenizing them in a 2 mL microcentrifuge tube  containing 250 μL of LB broth and two stainless steel beads  on ice. Collected tissues were homogenized at a frequency of 30 Hz for 20 seconds using a tissue homogenizer  before being centrifuged at 420 × g for 1 minute at 4 °C. Homogenates from each beetle were added into 180 μL of PBS and then serially diluted 1:10 to 1:105 , with host survival as a response variable, and with knockdown treatments and infection treatments as explanatory variables along with experimental replicate as a random factor (package ‘coxme’)  . When reP. entomophila bacterial load retrieved from the beetles, measured as the number of CFU obtained on agar plates, with a generalized linear model fitted for a negative binomial distribution  around the estimates of the optimal model. The differences were considered significant if two 95%CI error bars overlap by no more than about half of their length . Models with the lowest AIC - or lowest degree of complexity for an equivalent AIC - were retained as the optimal model. Models comprised within a delta AIC of two were considered equivalent .EGFP as a negative control, the results yielded by different knockdown treatments were not directly comparable. However, we are able to circumvent this experimental feature by comparing the effect sizes of the knockdowns relative to their respective control treatments, both for bacterial load and host survival. This approach allows for a robust comparison of the effects of single, double, and triple knockdowns of the TmAttacin genes expression between experiments, as shown in Zanchi et al. (2017) (EGFP) which is shown in the last section of the results.Since different experiments on single-, double-, or triple-knockdowns had a different amount of ds. (2017) . TherefoEGFP control within each experiment, and represents the relative risk of dying of the knockdown versus dsEGFP , obtained from the summary of the optimal model of each experiment, as a measure of effect size. The HR consists of the ratio of the hazard rates of the knockdown treatment to the respective dss dsEGFP .EGFP control, divided by the weighted pooled standard deviation for the two treatments , pairs of (TmAtt1a-Att2 and TmAtt1b-Att2), and all three (TmAtt1a-Att1b-Att2) AMP genes were silenced.The result of the induction experiment was consistent with previous study that shows that bacterial challenge led to a significant increase in transcription of controls . Based oTmAtt1a-treated females, we observed a consistent decrease of TmAtt1a mRNA transcript, ranging from 4-day to 14-day post-exposure. Accordingly, the 4-day RNAi treatment was selected as a time point for bacterial challenge  had no effect on expression level of either TmAtt1b or TmAtt2, similarly, TmAtt1b (3-day) knockdown did not influence transcript levels of TmAtt1a or TmAtt2. Based on the double-knockdown results, we altered the volume of representative genes to successfully silence all three genes. In triple-knockdown beetles we accounted for the reduced expression of TmAtt1a, TmAtt1b, and TmAtt2 after RNAi treatment of each gene separately. Therefore, we expected that 4-day post-exposure females would be optimal for challenging, which was confirmed via qRT-PCR  showed no death over 35 days, therefore we did not include them in the analysis, but presented them in the figures. We did not find an effect of knockdown treatment in P. entomophila injected beetles, since the survival of infected-dsTmAtt1a  was not significantly different from the control treatments   nor as a simple effect . CFU counts increased over 3 days   S17A.3.2.25.175 = 3.41, p = 0.18). However, it is important to note that the mortality in the PBS injected beetles is negligible across all knockdown treatments (only 3 dead beetles)  and infection treatments on beetle survival . We observed a strong decrease in survival of beetles after P. entomophila challenge  while no difference in survival of infected full control and infected dsEGFP was found .Both knockdown and infection treatment influenced beetle survival as simple effects . While the density of P. entomophila in dsEGFP-treated females increased between 1-day and 2-day post- infection, bacterial load of dsTmAtt1b-treated beetles showed higher densities starting from 1-day post infection. CFU counts of P. entomophila were similar between TmAtt1b knockdown treatment and control at 2-days post-infection. Interestingly, bacterial load of dsEGFP-treated females significantly decreased between 2-days and 3-days post-infection, whereas it remained similarly high between 2- and 3-days in dsTmAtt1b-treated beetles . Instead, both the knockdown and the infection treatments affect survival of beetles as simple effects. Again, TmAtt2-silenced treatments showed a decreased survival compared to the dsEGFP control , whereas the survival of dsEGFP beetles does not differ compared to full control  .Similarly, to the KD of TmAtt2-silenced beetles compared to the control . While CFU counts increased over 3 days in the control, as seen in the previous knockdowns, they already reached higher counts 1-day post-injection in dsTmAtt2-treated group compared to dsEGFP control. TmAtt2-silenced beetles increased and remained stable over the first two days post infection, whereas the control reached similar CFU counts at 3-days post challenge  nor as a simple effect . Only the infection treatment was retained in the best model, where the survival of adult females infected with P. entomophila was significantly lower  . dsEGFP-treated beetles showed the same dynamics as described above, the CFU count increased between 1-day and 2-days post-infection and remained stable between 2- and 3-days post-infection. Double-knockdown of TmAtt1a and TmAtt2 resulted in a higher number of CFU recovered from beetles starting from 1-day after challenge. Moreover, it reached a higher CFU count than the control at 2-days post infection and at 3-days decreased to a level similar to controls , the best model is one in which beetle survival is explained by both simple effects of the infection  and knockdown treatments . As expected, P. entomophila infection decreased beetle survival. However, the effect of the KD treatment does not come from a lower survival of TmAtt1b-Att2 compared to dsEGFP . It is caused by the fact that beetles with no injection of dsRNA outlived both dsEGFP  and dsTmAtt1b-Att2  . The CFU counts are similar at 1-day post-infection in both treatments but diverge at 2-days, where they reach higher concentrations in the KD. This effect persists at 3-days post-infection  display no mortality over the time course of this experiment. We further carried out the survival analysis on P. entomophila injected beetles only, however PBS-injected treatments are displayed in the figures. We detected that knockdown treatment significantly affects the survival of P. entomophila injected beetles . The triple knockdown of TmAtt1a, TmAtt1b, and TmAtt2 by RNAi reduced the survival of females following P. entomophila infection  while survival of dsEGFP beetles does not differ compared to full control beetles  , however, the CFU counts are explained by both time  and knockdown treatment  as simple effects. P. entomophila load increased initially (1-day and 2-days post-infection) and remained stable at the last day in both treatments. CFU counts were overall higher in TmAtt1a-Att1b-Att2-silenced beetles than in the control . This is not the case at 2-  and 3-days  post-infection.The differential dynamics of the effects of KD on bacterial load was not reflected in the survival curves, as shown by the hazard ratios (HR)  but of lesser magnitude compared to the single knockdown of TmAtt2 and resulted in no detectable changes in host survival. In this regard, the increased bacterial burden might be responsible for the early host mortality, whereas a later decrease in bacterial load did not influence mortality. Similarly, double-knockdown of TmAtt1b-Att2 could be responsible for the drastic increase in P. entomophila colonization which on the contrary does not result in an increase in mortality rate. Therefore, both double knockdown treatments increased bacterial loads at later time points after infection but did not influence host survival.In the case of the double knockdowns, we observed that dsP. entomophila infection resulted in the death of the majority of triple-knockdown females within 3 days . Knockdown of TmAtt1a in combination with either TmAtt1b or TmAtt2 is inefficient (TmAtt1a-Att1b or TmAtt1a-Att2). This indicates an antagonistic effect of TmAtt1a on TmAtt1b and TmAtt2, however, the difference between the effect sizes of the knockdown treatments is not significant. Therefore, the datasets of the single- and double-knockdowns do not allow us to decide whether this pattern is the result of antagonism, redundancy, synergy, or an additive effect between the three TmAttacins in the context of a double-knockdown.Following this reasoning, we focused on the effects of the different knockdown treatments on early bacterial load (1-day post-infection) and on host survival in response to P.entomophila load is greatest at 1-day post-infection. This observation seems to rule out the possibility of an antagonistic interaction and suggests an additive effect of these three members of the TmAttacin family. The effect of the triple-knockdown, although larger, is not significantly different from the effect of the single knockdown of TmAtt1b, which could imply that the effects observed with the triple-knockdown treatment could be due exclusively to TmAtt1b. However, this seems unlikely because we do not observe a survival phenotype with the double-knockdown of TmAtt1b-Att2.The effect of triple-knockdown on host survival and TmAtt1a + TmAtt1b + TmAtt2 = 2.39 vs. 2.23 for the triple-knockdown). This is not the case for the double-knockdowns, where, on the contrary, the effect sizes of the double- knockdowns are close to the effect sizes of the corresponding single-knockdowns minus each other . This suggests that the effects of single-, double-, and triple-knockdowns of our three AMPs alter the nature of the interactions between them.A closer look at the value of the effect sizes on the CFU counts at 1-day post-infection shows that the effect size of the triple-knockdown on the CFU counts is close to the effect size of the three single knockdowns combined (Hedge’s g D. melanogaster. This highlighted additive or synergistic anti-Gram-negative and antifungal activities between AMPs as well as in vivo specific AMP-pathogen interactions (Tenebrio molitor. Our data also concur with in vitro experiments carried by Yu et al. (in vivo between AMPs of different classes in Zanchi et al. (T. molitor is more efficiently controlled by a combination of AMPs than by single ones (in vivo is still very limited, as studies have either knocked out groups of AMPs, for example (in vivo is, as determining synergy for example is conceptually and empirically challenging (These two possibilities were previously addressed by Hanson et al. , who knoractions , 13. In ractions . Our datu et al. , who repgle ones . Overall example  knocked  example . The appllenging .TmAtt1a and TmAtt1b by overexpression of one other of our focal AMPs. Nevertheless, we cannot exclude that other members of the Attacin family or of other AMP families would be upregulated following the KD of one or several in vivo. This would add another level of interactions. In spite of this, our work demonstrates, that at least within the Attacin group, when AMPs are knocked down, the overall Attacin response in reduced, rendering beetles more susceptible to infection.To our knowledge, whether the depletion of transcripts of some AMP genes could be compensated by the upregulation of others has never been investigated, which would be an important direction for future research. We did not find evidence for compensation of the depletion of transcripts of The original contributions presented in the study are included in the article/Conceptualization: MK, CZ and JR. MK designed and performed the experiments. MK and CZ analyzed the data and wrote the manuscript. JR revised the manuscript and procured reagents and materials. All authors contributed to the article and approved the submitted version.